Amplified Fragment Length Polymorphism Fingerprinting Is an Effective Technique To Distinguish Streptococcus pneumoniae from Other Streptococci and an Efficient Alternative to Pulsed-Field Gel Electrophoresis for Molecular Typing of Pneumococci

AbstractObjective:To compare molecular typing by amplified fragment-length polymorphism (AFLP) analysis with pulsed-field gel electrophoresis (PFGE) with respect to the ability to differentiate between epidemiologically related and unrelated isolates of common nosocomial pathogens recovered during a period of endemicity.Design:Retrospective laboratory analysis.Setting:Tertiary-care institution.Methods:17 isolates ofAcinetobacter baumannii,22 isolates ofPseudomonas aeruginosa,and 22 vancomycin-resistantEnterococcus faecium(VRE) were typed by both methods.Results:AFLP generated comparable results to PFGE forA baumanniiandP aeruginosaisolates; both methods identified epidemiologically related and unrelated isolates. However, strain typing of VRE isolates produced discordant results between the two methods. PFGE identified 10 different strain types and differentiated between all epidemiologically related and unrelated isolates. In contrast, AFLP generated only five different strain types, three of which contained both epidemiologically related and unrelated isolates.Conclusion:Molecular typing by AFLP is comparable to PFGE forA baumanniiandP aeruginosaisolates. For VRE isolates, however, PFGE remains the method of choice.

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Evaluation of Fluorescence-Based Amplified Fragment Length Polymorphism Analysis for Molecular Typing in Hospital Epidemiology: Comparison with Pulsed-Field Gel Electrophoresis for Typing Strains of Vancomycin-Resistant Enterococcus faecium

Journal of Clinical Microbiology ◽

10.1128/jcm.38.11.4058-4065.2000 ◽

2000 ◽

Vol 38 (11) ◽

pp. 4058-4065 ◽

Cited By ~ 23

Author(s):

Nick A. Antonishyn ◽

Ryan R. McDonald ◽

Edward L. Chan ◽

Greg Horsman ◽

Carla E. Woodmansee ◽

...

Keyword(s):

Length Polymorphism ◽

Fragment Length ◽

Molecular Typing ◽

Gel Electrophoresis ◽

Fragment Length Polymorphism ◽

Amplified Fragment Length Polymorphism ◽

Pulsed Field Gel Electrophoresis ◽

Restriction Fragments ◽

Pulsed Field ◽

Vancomycin Resistant

Fluorescence-based amplified fragment length polymorphism (fbAFLP) is a novel assay based on the fluorescent analysis of an amplified subset of restriction fragments. The fbAFLP assay involves the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The ligation of adapters with primer-specific sites coupled with primers containing selective nucleotides allowed the full potential of PCR to be realized while maintaining the advantages of restriction endonuclease analysis. Fluorescence-based fragment analysis with polyacrylamide gel electrophoresis provides the accurate band sizing required for homology assessment. The large number of phylogenetically informative characters obtained by fbAFLP is well suited for cluster analysis and database development. The method demonstrated excellent reproducibility and ease of performance and interpretation. We typed 30 epidemiologically well-characterized isolates of vancomycin-resistant enterococci from an outbreak in a university hospital by fbAFLP. Clustering of fbAFLP data matched epidemiological, microbiological, and pulsed-field gel electrophoresis data. This study demonstrates the unprecedented utility of fbAFLP for epidemiological investigation. Future developments in standardization and automation will set fbAFLP as the “gold standard” for molecular typing in epidemiology.

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